independent apoptosis in PC12 cells

Food and Chemical Toxicology 134 (2019) 110835

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Carvacrol inhibits cadmium toxicity through combating against caspase dependent/independent apoptosis in PC12 cells

T

Subrata Banika, Mahmuda Aktera, Serene Ezra Corpus Bondada, Takeshi Saitob, Toshiyuki Hosokawac, Masaaki Kurasakia,d,∗ a

Graduate School of Environmental Science, Hokkaido University, 060-0810, Sapporo, Japan Faculty of Health Sciences, Hokkaido University, 060-0812, Sapporo, Japan c Institute for the Advancement of Higher Education, Hokkaido University, 060-0810, Sapporo, Japan d Faculty of Environmental Earth Science, Hokkaido University, 060-0817, Sapporo, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Food antioxidants Oxidative stress Glutathione Pro-survival proteins Cytochrome c Apoptosis inducing factor

Carvacrol is a monoterpenic phenol found in essential oils, is considered a safe food additive, and possesses various therapeutic properties. Numerous studies have also deciphered the protective role of carvacrol on various cytotoxicities. We clarify the effects of carvacrol on cadmium-induced apoptosis in PC12 cells. Carvacrol while co-exposed with cadmium for 48 h raised PC12 cell viability in comparison to only cadmium exposed group. The co-exposure increased the cellular glutathione levels and promoted the expression of glutathione reductase. The magnitude of DNA fragmentation caused by cadmium was also ameliorated by carvacrol. Flow cytometry exhibited the apoptosis rate augmented by cadmium was reduced by carvacrol. Western blotting revealed that cadmium and carvacrol co-exposure alleviated the cadmium-induced down-regulations of mammalian target of rapamycin (mTOR), protein kinase B (Akt), nuclear factor kappa-light-chain-enhancer of activated B cells (NFКB), extracellular signal-regulated kinase-1 (ERK-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions. The co-exposure also reversed action of cadmium by suppressing the cleavage of caspase 3 and reducing the cytosolic levels of cytochrome c and apoptosis inducing factor (AIF). Moreover, carvacrol upon co-exposure significantly increased the intracellular metallothionein content. In conclusion, carvacrol strongly reduced cadmium-triggered oxidative stress and caspase-dependent and caspase-independent apoptosis in PC12 cells.

1. Introduction Cadmium (Cd2+), a heavy metal used in many industrial and household products, has become prioritized due to its emergence as a widespread environmental pollutant and a biological toxicant. Concern over Cd2+ is due to its extremely high toxicity, a long half-life in humans and being a causative agent for many diseases and disorders upon acute or chronic exposure (Järup and Åkesson, 2009; Jiang et al., 2014). Evidences support that Cd2+ exposure is associated with a variety of ailments including renal tubular dysfunction (Nishijo et al., 2006), osteoporosis (James and Meliker, 2013), hepatotoxicity (Dudley et al., 1982), cardiovascular diseases (Tellez-Plaza et al., 2013) and neurotoxicity (Wang and Du, 2013). Following absorption and distribution, Cd2+ affects cellular metabolic processes resulting to alterations in proliferation and differentiation. Cd2+ causes cellular injury through exerting oxidative stress (Patra et al., 2011), promoting DNA

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damage (Badisa et al., 2007), altering transport pathways (Kerkhove et al., 2010) and impairing mitochondrial membrane potential leading to apoptosis (Mao et al., 2011; Jiang et al., 2014). The landscape for Cd2+-triggered death mechanisms have been studied extensively in various cell lines. However, the death mechanisms differ depending on cell type, Cd2+ concentration and duration of exposure. Despite many debates and complexities of mechanisms, previous reports detailed that in vitro cells frequently go through an apoptotic death at low to moderate (e.g., 0.1–10 μM) Cd2+ concentrations and undergo necrosis at high (> 50 μM) concentrations (Templeton and Liu, 2010). Numerous cell systems showed that diverse signaling pathways have been involved in Cd2+-induced apoptosis including mitochondrial (Rahman et al., 2017), extrinsic (Liu et al., 2016a) and caspase-independent (Liu and Templeton, 2008) pathways. But, a rise in reactive oxygen species (ROS) levels, increased lipid peroxidation, alterations in antioxidant defense system, and stimulation of metallothionein formation are the

Corresponding author. Faculty of Environmental Earth Science, Hokkaido University, Sapporo, 060-0810, Japan. E-mail address: [email protected] (M. Kurasaki).

https://doi.org/10.1016/j.fct.2019.110835 Received 10 August 2019; Received in revised form 19 September 2019; Accepted 21 September 2019 Available online 25 September 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved.

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Abbreviations AIF Akt ANOVA CVC DTNB DTT ECL EGTA ELISA ERK-1 FBS GR GSH

HEPES HRP LDH MT mTOR NaF NFКB

apoptosis inducing factor protein kinase B single-factor analysis of variance carvacrol 5, 5′-dithiobis-2-nitrobenzoic acid dithiothreitol enhanced chemiluminescence ethylene glycol tetraacetic acid enzyme-linked immunosorbent assay extracellular signal-regulated kinase-1 fetal bovine serum glutathione reductase glutathione

Nrf2 PBS PCR PMSF ROS SEM

common phenomena cells follow upon Cd2+-induced cytotoxicity, irrespective of apoptotic pathway(s) (Stohs et al., 2000; Sarkar et al., 1998). Carvacrol (5-isopropyl-2-methyl-phenol) (CVC) is, a monoterpenoid phenol predominantly found as a natural constituent of various essential oils of Labiateae family plant species such as oregano, thyme and pepperwort (Noshy et al., 2018; Kirimer et al., 1995). It is generally considered as a food additive and flavoring agent (Zotti et al., 2013). Recently edible and non-edible CVC-based films have become very useful components in antimicrobial food packaging (Ramos et al., 2016). Numerous studies have reported the diverse biological and therapeutic properties of CVC including antimicrobial (Xu et al., 2008), antioxidant (Beena et al., 2013; Aristatile et al., 2009), anti-inflammatory (Landa et al., 2009), anti-carcinogenic (Karkabounas et al., 2006) and neuromodulatory (Zotti et al., 2013) activities. Cumulative evidences from in vivo and in vitro studies have already confirmed the protective role of CVC against various toxicant-induced oxidative stress and apoptosis in various organs and cell lines (Noshy et al., 2018; Aristatile et al., 2009; Wang et al., 2017; Samarghandian et al., 2016; Palabiyik et al., 2016). Another study also demonstrated that CVC can also alleviate Fe2+-induced oxidative stress and apoptosis in SH-SY5Y cells (Cui et al., 2015). However, no report becomes available with regards to the potential protective effects of CVC on Cd2+-induced toxicity in cell system with bio-molecular mechanistic clarifications. Therefore, we hypothesized that CVC can induce protective effects against Cd2+-induced cytotoxicity in cultured cells. Experiments were conducted to evaluate whether CVC has ameliorative effects against the cytotoxicity, oxidative stress and apoptosis caused by Cd2+ on PC12 cells. This study is assumed to be the first approach which would elucidate the abovementioned properties of CVC in a model cell line.

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid horseradish peroxidase lactate dehydorogenase metallothionein mammalian target of rapamycin sodium fluoride nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor erythroid 2-related factor 2 phosphate buffered saline polymerase chain reaction phenylmethylsulfonyl fluoride reactive oxygen species standard error of mean

Biotechnology), extracellular signal-regulated kinase 1 (ERK1) (61003c, BD Transduction Laboratories), nuclear factor erythroid 2related factor 2 (Nrf2) (PM069, MBL; Japan), caspase 3 (GTX110543, GeneTEX), and apoptosis inducing factor (AIF) (cat# 551429, BD Biosciences) were also procured. The Cytochrome c Release Apoptosis Kit (Q1A87-1KIT) was purchased from CalbiochemR. Anti-mouse IgG (H + L) horseradish peroxidase (HRP) conjugate (W4021) and antirabbit IgG (H + L) horseradish peroxidase (HRP) conjugate (W4011) were bought from Promega Corporation (Madison, WI, USA). Enhanced chemiluminescence (ECL) western blotting detection reagent (Amersham Pharmacia Biotech., Buckinghamshire, England) and trypan blue (0.4%) solution (Bio-Rad, Hercules, CA, USA) were purchased. The AnnexinA5-FITC flowcytometry kit (cat# IM2375) was procured from Beckman Coulter (Marseille Cedex9, France) and metallothionein (MT) enzyme-linked immunosorbant assay (ELISA) kit (Lot# 160361) was collected from Frontier Science Co. Ltd. (Hokkaido, Japan). All other chemicals used were of analytical grade. 2.2. Cell culture and treatments PC12 cells were cultured on 25 cm2 cell culture flasks in DMEM supplemented with 10% fetal bovine serum (FBS) in a dehumidified incubator at 37 °C with 5% CO2. After a 24 h pre-incubation, the cells were exposed to CdCl2 (Cd2+), or to CVC, or to both for 48 h. The exposure concentrations of CVC was first chosen as 0, 50, 100, 200 and 400 μM. But for further experimentation, the concentrations of Cd2+ and CVC used were selected as 10 μM and 100 μM, respectively. 2.3. Cell viability Trypan blue exclusion test was used for the assessment of cell viability. PC12 cells were seeded at a density of about 1×105/flask and pre-incubated for 24 h until logarithmic growth phase was reached. The cells were then exposed with different concentrations of CVC (0, 50, 100, 200 and 400 μM) and Cd2+ (10 μM), separately; Co-exposure was done to cells using different non-toxic concentrations of CVC (0, 50, 100 and 200 μM) and Cd2+ (10 μM). After treatments and co-treatments, the cells were incubated for 48 h. Then, cells were subsequently collected and stained with 0.2% trypan blue in 1 × phosphate-buffered solution (PBS). The number of trypan blue-stained cells and total cells were counted using a cell counter (TC10™ Automated Cell Counter, Bio-Rad). Cell viability was expressed as percentage (%) of the trypan bluestained cells.

2. Materials and methods 2.1. Materials PC12 cells were obtained from American Type Culture Collection (USA and Canada). Dulbecco's modified Eagle's medium (DMEM), ribonuclease A (RNase), ethidium bromide, and peroxidase-conjugated avidin were bought from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Biosera (Kansas City, MO, USA). High pure polymerase chain reaction (PCR) product purification kit and proteinase K were acquired from Roche Diagnostics (Mannheim, Germany). Polyclonal antibodies against β-actin (cat# 4967), mammalian target of rapamycin (mTOR) (cat# 2972), cleaved caspase 3 (cat# 9661) and protein kinase B (Akt) (cat# 4691) were purchased from Cell Signaling Technology. Polyclonal antibodies against glutathione reductase (GR) (ab16801, Abcam), nuclear factor kappa-lightchain-enhancer of activated B cells (NFκB) (sc-109, Santa Cruz

2.4. Lactate dehydrogenase (LDH) activity assay Cytotoxicity-derived cell membrane disintegration levels were assessed by measuring LDH activity in the cell treatment medium using a 2

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DNA density to the fragmented DNA density. This experiment was repeated for at least three times to ensure reproducibility.

nonradioactive cytotoxicity assay kit as described by Rahman et al. (2018). PC12 cells were exposed to Cd2+ (10 μM) or CVC (100 μM), or Cd2+ (10 μM) + CVC (100 μM) for 48 h. After exposure period, 50 μL of the culture medium was contained in a 1.5 mL tube and subsequently, 50 μL of substrate mixture (containing tetrazolium salts) was added to the tube. After incubating at room temperature for 30-min, 50 μL of stop solution was added. The absorbance at 490 nm was measured using an iMark™ microplate reader (BioRad; Hercules, CA, USA) to determine the amount of formazan dye produced. LDH activity was expressed as “LDH activity/1 × 106 cells”. This experiment was repeated at least 3 times for ensuring biological and statistical reproducibility.

2.8. Detection of apoptosis rate by flow cytometry The apoptosis rate of PC12 cells was detected by flow cytometric analysis. PC12 cells were harvested after 48 h exposure to 10 μM Cd2+, 100 μM CVC or to combined 10 μM Cd2+ and 100 μM CVC. Then cells were washed with 1 × PBS and 400 μL ice-cold 1 × binding buffer was subsequently added to the cells. Afterwards, 5 μL of annexin A5-FITC solution and 2.5 μL of propidium iodide (PI) were added to the cells and the resulting solution was kept for 10 min in the dark. Finally, samples were analyzed using a BD FACSVerse™ Flowcytometer. The experiment was conducted at least in triplicates for biological and statistical reproducibility.

2.5. Measurement of intracellular glutathione (GSH) levels Intracellular free-SH levels were determined following the methods previously described by Kihara et al. (2012) and Rahman et al. (2018). PC12 cells were harvested after 48 h exposure to Cd2+ (10 μM), CVC (100 μM) or Cd2+ (10 μM) + CVC (100 μM). The cells were then washed with 1 × PBS. A lysis buffer at an amount of 150 μL was added to the cells and incubated at room temperature for 10 min. Two freezethaw sonication cycles were executed in order to rupture the cell membranes. The resulting solution was centrifuged at 1500 rpm for 10 min to collect the supernatant. Then, the protein contents were measured spectrophotometrically using a protein assay dye reagent (Bio-Rad; Hercules, CA, USA). GSH levels were measured using 2.5 mM 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB, pH 7). DTNB was added to the cell lysate at a concentration of 200 μM, and the absorbance at 412 nm was measured with a DU-65 spectrophotometer (Beckman, CA, USA). The free–SH concentration was determined by using a molecular coefficient factor of 13,600 per cell number (1 × 105). The experiments were conducted at least triplicate to achieve reproducibility.

2.9. Western blot analysis for the determination of protein expressions Western blot analysis was accomplished for the determination of protein expressions in PC12 cells 48 h after exposure to the aforementioned Cd2+ and/or CVC concentrations. Cells were harvested and washed by suspending in ice-cold 1 × PBS, then centrifuged at 1500 rpm for 10 min. After removing the supernatant the cells were resuspended in 150 μL of lysis buffer (consisting of 2 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 mM NaCl, 10 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 0.1 mM Na2MoO4, 5 mM 2-glycerophosphorate, 10 mM MgCl2, 2 mM dithiothreitol (DTT), 50 mM sodium fluoride (NaF), and 1% Triton X-100) for total protein extraction. The mixture was allowed to stand on ice for 10 min and then cells were disrupted by two cycles of sonication followed by centrifugation at 1500 rpm for 10 min to collect the lysate containing the total cellular protein. On the other hand, the cytosolic protein fractions were extracted using the cytosolic lysis buffer provided in the ‘cytochrome c release apoptosis kit (Q1A87-1KIT)’. The concentration of extracted protein in the lysates was determined spectrophotometrically using a protein assay dye reagent (BioRad, Hercules, CA, USA). Thereafter, an equal amount (20 μg) of each protein sample was separated by 12.5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresed proteins were then transferred into a nitrocellulose membrane using a semi-dry blotting system (type- AE6678; ATTO, Tokyo, Japan). The membranes were incubated overnight at 4 °C in 5% skimmed milk as a blocking agent. After blocking, each membrane was washed three times with 0.1% Tween buffer and incubated with the desired primary and secondary antibodies. After washing with 0.1% Tween buffer, the protein band on the nitrocellulose membrane was visualized using enhanced chemiluminescence, and analyzed using a ChemiDoc XRS (Bio-Rad, USA). Finally, the band intensities of the targeted proteins were expressed as the ratio to the intensity of β-actin. Each experiment was conducted at least in triplicates to ensure reproducibility.

2.6. Isolation of the genomic DNA The genomic DNA of PC12 cells was isolated using high pure PCR template preparation kit following the manufacturer's instructions. After treatment with Cd2+ (10 μM), CVC (100 μM) or Cd2+ (10 μM) + CVC (100 μM) cells were incubated for 48 h. Then, the cells were harvested using a scraper, and centrifuged at 1500 rpm for 5 min to remove the supernatant. Afterwards, the cells were washed with 1 × PBS and centrifuged at 1500 rpm for 5 min. The procedure for isolation of the genomic DNA was then performed. The isolated DNA was incubated with RNase (10 μg/mL) for 15 min at 37 °C, followed by addition of 100% ethanol and 3M NaOAc buffer (pH 4.5) were added. The solution was allowed to stand overnight at −20 °C for DNA precipitation. The following day, precipitated DNA was centrifuged at 15,000 rpm for 7 min, and for another 3 min after being washed with 70% ethanol. The obtained DNA was dried and solved with 50 μL 1 × Tris/Borate/Ethylenediaminetetraacetic acid (TBE), followed by the measurement of DNA concentration using a GeneQuant (GE Heath Care; South East England, UK). Finally, DNA concentrations were equalized for all samples by adding 1×TBE solution. 2.7. Agarose gel electrophoresis of genomic DNA

2.10. Intracellular,Cd2+ accumulation in PC12 cells The extracted genomic DNA in PC12 cells was subjected to agarose gel electrophoresis to assess the fragmentation levels. DNA (5 μg) was mixed with loading dye and electrophoresed on a 1.5% agarose gel for 40 min at 100 V, using a submarine-type electrophoresis system (Mupid-ex, Advance, Tokyo, Japan). Following electrophoresis, the gel was soaked in ethidium bromide solution for 5–10 min. DNA fragmentation was visualized under UV illumination using a ChemiDoc XRS (Bio-Rad; Hercules, CA, USA). The fluorescence intensity of fragmented DNA was analyzed using a software named “Quantity One”. The amount of intact DNA was expressed as the intensity ratio of the total

The PC12 cells were incubated for 48 h after being exposed to 10 μM Cd2+ and/or 100 μM CVC. Following harvesting and washing with 1 × PBS cells were digested with 1 M nitric acid at 70 °C for 1 h. Then, the digested samples were filtered through a 0.22 μM pore membrane and diluted using deionized water to prepare for measurement. Intracellular cadmium contents were measured by using an ICPE-9000 inductively coupled plasma mass spectrometry (ICP-MS) (Shimadzu; Kyoto, Japan). To confirm the reproducibility, this experiment was carried out in triplicate. 3

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2013 program.

2.11. Determination of metallothionein (MT) content PC12 cells were exposed to 10 μM Cd2+ and/or 100 μM CVC for 48 h, harvested, and washed twice with 1 × PBS. The protein from the cells was extracted and the total protein content was measured spectrophotometrically using a protein assay dye reagent (Bio-Rad, Hercules, CA USA). The MT content in the cell lysate was measured by ELISA kit following manufacturer's protocol. The provided 96 well microtiter plate was first washed with PBS solution followed by the addition of 50 μL of standard or sample solution in each well. After adding 50 μL first antibody in each well, the microtiter plate was incubated at room temperature for 1 h. Then, each of the wells was washed thrice with 350 μL washing buffer and afterwards, 100 μL of second antibody was added. Following 1 h incubation at room temperature wells were washed and 100 μL of substrate mixture was subsequently added to each well while keeping the set-up in the dark. The reaction was stopped by adding 50 μL of stop solution. Finally, absorbance of the solution was measured at 450 nm in an ELISA reader (ng/ mg of protein) and the concentration was calculated from a standard curve of MT protein.

3. Results 3.1. Effects of Cd2+ and CVC on the viability of PC12 cells To investigate the toxicity of CVC, PC12 cells were exposed to 50, 100, 200 and 400 μM of CVC for 48 h. Cells were also exposed to 10 μM of Cd2+ and combined Cd2+ and CVC. Trypan blue staining method was performed to assess the viability of PC12 cells. The results showed that the treatments of CVC up-to 200 μM did not affect the cell viability (Fig. 1A), indicating that the CVC concentrations used in this study were not toxic. On the other hand, the decrease in the PC12 cell viability caused by 10 μM Cd2+ was significantly (p < 0.05) prevented by CVC (100 or 200 μM) (Fig. 1B). Therefore, it was proposed from the cell viability results that CVC (100 or 200 μM) exerted cytoprotective effects against Cd2+ (10 μM)-induced cell death. The results were used as basis of choosing 10 μM Cd2+ and 100 μM CVC for further experimentation. 3.2. CVC reduces Cd2+ induced-LDH leakage

2.12. Statistical analysis

The soluble cytosolic enzyme LDH released in the cell culture media upon plasma membrane disintegration is one of the important indicators of toxicant-induced cell death. The LDH activity was measured in PC12 cell culture media after 48 h exposure to Cd2+ (10 μM) and/or CVC (100 μM). As shown in Fig. 2, compared to the control, Cd2+ (10 μM) exposure noticeably (p < 0.05) increased the LDH activity in

Data were represented as the mean ± standard error of mean (SEM); p < 0.05 was considered to indicate significance level. Analysis of statistical significance was achieved by using single-factor analysis of variance (ANOVA) followed by unpaired Student's t-test in MS Excel

Fig. 1. Viability (%) of PC12 cells using trypan blue exclusion method. (A) PC12 cells exposed with 50, 100, 200 and 400 μM CVC for 48 h. (B) PC12 cells exposed/coexposed with 10 μM Cd2+and 50, 100, and 200 μM CVC for 48 h. Error bars indicate mean ± S.E.M. (n = 4 for (A) and 3 for (B)), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively. 4

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Fig. 2. LDH activity in the culture medium of PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h measured by a nonradioactive cytotoxicity assay kit. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively.

CVC showed antagonizing role on Cd2+-induced oxidative damage.

the culture medium. On the other hand, no significant change in LDH activity was observed 48 h after CVC (100 μM) exposure. The combined exposure of Cd2+ (10 μM) and CVC (100 μM) significantly (p < 0.05) reduced the LDH activity in the medium compared to that only Cd2+ (10 μM) exposed group.

3.4. CVC alleviates Cd2+-induced genomic DNA damage Genomic DNA damage is one of the major biological outcome of cadmium toxicity and is also a prominent route of cell inactivation in apoptosis (Roos and Kaina, 2006). In this study, agarose gel electrophoresis results exhibited that the amount of intact genomic DNA in PC12 cells was severely diminished after 48 h of Cd2+ (10 μM) exposure (Fig. 4A). On the other hand, it became visible that the exposure to CVC (100 μM) had no effect on the amount of intact genomic DNA compared to that of the untreated control cells. Cd2+ (10 μM)-induced DNA fragmentation was lessened in the case of cells exposed to combined Cd2+ (10 μM) and CVC (100 μM). In the line with this, we found a significant decrease in DNA band density in Cd2+ (10 μM) exposed cells, while the DNA band density for cells co-exposed with Cd2+ (10 μM) and CVC (100 μM) was markedly (p < 0.05) higher in comparison to only Cd2+ (10 μM) treated group (Fig. 4B). Thus, the coexposed cell group possessed a comparatively reduced DNA damage.

3.3. Effects of Cd2+and CVC on the intracellular GSH levels and GR expression GSH is the most abundant intracellular non-protein thiol which acts as an antioxidative defense system and removes oxidative stress generating electrophiles to maintain the redox homeostasis (Du et al., 2009). We exposed PC12 cells to Cd2+ (10 μM) and/or CVC (100 μM) for 48 h, and then measured intracellular GSH levels. A significant (p < 0.05) decrease in intracellular GSH content was found in the cells exposed to Cd2+ (10 μM), which was markedly (p < 0.05) increased in the cells with combined Cd2+ (10 μM) and CVC (100 μM) (Fig. 3A). However, no change in GSH level was found in cells exposed to CVC (100 μM) only. Thus, it can be suggested that CVC ameliorated oxidative stress posed by Cd2+ in PC12 cells. Additionally, Western blot analysis showed that expression of GR, the enzyme converting GSSG to GSH, supported the results found for GSH levels (Fig. 3B). Therefore,

Fig. 3. Oxidative stress markers in PC12 cells exposed/co-exposed with Cd2+ and CVC. (A) Intracellular glutathione (GSH) level in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h measured using DTNB. (B) GR expressed in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h analyzed by western blotting. Error bars indicate mean ± S.E.M. (n = 4), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+exposed group respectively. 5

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3.5. CVC decreases the rate of Cd2+induced apoptosis in PC12 cells To determine the rate of apoptosis, PC12 cells were exposed to Cd2+ (10 μM) and/or CVC (100 μM) for 48 h. Then, the apoptotic rate was determined by annexin-V-FITC/PI assay using flow cytometry analysis (Fig. 5A). As shown in Fig. 5B, more than 54% of the Cd2+ (10 μM) exposed cells were in either early (in lower left (LL) quadrant) or late (in upper right (UR) quadrant) apoptotic condition. Although only CVC (100 μM) exposure did not change the apoptosis rate compared to that of the control group (4.65%), Cd2+ (10 μM) and CVC (100 μM) co-exposure markedly (p < 0.05) diminished the apoptosis rate to about 26%. These results suggest that CVC strongly reduced Cd2+-provoked apoptosis in PC12 cells. 3.6. Effects of Cd2+ and CVC on the expressions of pro-survival proteins We analyzed the expression of protein factors crucial for cell metabolism, proliferation, and survival in PC12 cells after exposure to Cd2+ (10 μM), or CVC (100 μM), or co-exposing with both Cd2+ (10 μM) and CVC (100 μM). The Western blot analyses images for the expressions of mTOR, Akt and NFκB were shown in Fig. 6A. Results exhibited that only Cd2+ (10 μM) exposure significantly (p < 0.05) down-regulated the expressions of mTOR, Akt and NFκB in comparison to the control. Compared to Cd2+ (10 μM) exposed group, all of these pro-survival proteins were evidently (p < 0.05) up-regulated in PC12 cells co-exposed to Cd2+ (10 μM) and CVC (100 μM) (Fig. 6B). Thus, the above results support that CVC (100 μM) could enhance the survival rate and protect PC12 cells from Cd2+ (10 μM)-induced oxidative stress and apoptosis.

Fig. 4. Agarose gel electrophoresis of genomic DNA extracted from PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h. (A) A sample electrophoresis image showing the content of intact and fragmented genomic DNA of PC12 cells exposed/co-exposed with Cd2+ and CVC. (B) Relative band intensity of intact DNA for fragmentation detection. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively.

3.7. CVC enhances the expressions of ERK1 and Nrf2 suppressed by Cd2+ ERK1, a member of mitogen-activated protein kinase (MAPK) protein family, is generally considered as a regulator of many pro-survival and anti-apoptotic proteins. On the other hand, Nrf2 is a transcription factor that regulates the expressions of many antioxidant enzymes. In Fig. 7A, the representative Western blot images of the expressions of ERK1 and Nrf2 in PC12 cells showed that the expressions of both ERK1

Fig. 5. The apoptotic rate of PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h analyzed by flow cytometry. (A) A representative experimental result of flow cytometry followed by annexin V-fluorescein isothiocyanate (FITC) and PI staining of PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. (B) The apoptotic rate of PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h, calculated and analyzed using early apoptosis (LR) and late apoptosis (UR). Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively. 6

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Fig. 6. Western blot analysis for the expression of pro-survival proteins in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h. (A) The representative images (cropped) of immunoblotting for the expressions of mTOR, Akt and NFκB in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. (B) Relative density for the expression bands of mTOR, Akt and NFκB to β-actin in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively.

3.9. Effects of CVC on Cd2+-induced release of cytochrome c and AIF from mitochondria into the cytosol

and Nrf2 became significantly (p < 0.05) lessened due to the exposure to Cd2+ (10 μM) for 48 h. But, the band for co-exposed group was much more visible. The quantified expression levels of ERK1 and Nrf2 represented in Fig. 7B imply that upon co-exposure CVC (100 μM) significantly (p < 0.05) alleviated the Cd2+ (10 μM)-induced reduction of ERK1 and Nrf2 protein expressions.

The release of the hallmark protein cytochrome c, from the mitochondria into the cytosol, is essential to initiate the caspase cascade reaction leading to apoptosis. On the other hand, the mitochondrial membrane protein, AIF takes part in chromatin condensation and DNA fragmentation; and is translocated to the nucleus via cytosol during apoptosis. The results show a significant (p < 0.05) increase in cytosolic cytochrome c and AIF after exposing PC12 cells with Cd2+ (10 μM), which became considerably (p < 0.05) lesser for Cd2+ (10 μM) and CVC (100 μM) co-exposed groups (Fig. 9A and B).

3.8. Effects of CVC on Cd2+-induced cleavage of caspase 3 The activation of caspase 3 by cleavage is the final event in the caspase cascade reactions in apoptotic cellular execution. PC12 cells were exposed to Cd2+ (10 μM), or CVC (100 μM), or both Cd2+ (10 μM) and CVC (100 μM) for 48 h. We found a substantial (p < 0.05) uplift in cleaved caspase 3 level in Cd2+ (10 μM) exposed cell group. Whereas, this level of cleaved caspase 3 was found to be lowered in cells exposed to combined Cd2+ (10 μM) and CVC (100 μM) (Fig. 8A and B). On the other hand, the expression of caspase 3 in Cd2+ (10 μM) exposed cells became down-regulated significantly but showed no difference with coexposed cell group. Therefore, from the results it was ascertain that CVC saves PC12 cells from Cd2+ (10 μM)-induced apoptosis by hindering the cleavage of caspase 3.

3.10. Effects of CVC on Cd2+ uptake and MT expressions in PC12 cells After exposing cells to Cd2+ (10 μM) and/or CVC (100 μM) for 48 h, the Cd2+ uptake by PC12 cells was measured using an ICP-MS. Although not significant, the Cd2+ uptake was higher in cells co-exposed to Cd2+ (10 μM) and CVC (100 μM) (Fig. 10) in comparison to cells exposed to Cd2+ (10 μM) only. Furthermore, we measured the MT expressions in the cells to explain the phenomenon of Cd2+ uptake increase in the co-exposed group. Fig. 11 shows that the expression of

Fig. 7. Western blot analysis for the expression of ERK-1 and Nrf2 in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h. (A) The representative images (cropped) of immunoblotting for the expressions of ERK-1 and Nrf2 in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. (B) Relative density for the expression bands of ERK-1 and Nrf2 to β-actin in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively. 7

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Fig. 8. Western blot analysis for the expression of caspase 3 and cleaved caspase 3 (c. caspase 3) in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h. (A) The representative images (cropped) of immunoblotting for the expressions of caspase 3 and c. caspase 3 in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. (B) Relative density for the expression bands of caspase 3 and c. caspase 3 to β-actin in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group respectively.

macromolecules and ultimately leading to apoptosis (Hu et al., 2015; Oh and Lim, 2006; Zhou et al., 2009; Chatterjee et al., 2009). In mammalian cells GSH plays a key role in combating ROS. ROS also interacts with classical antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and GR. The efficiency of GSH to regenerate depends on the redox state of the glutathione disulphide-glutathione couple (GSSG/2GSH) (Nemmiche, 2017). In line with this, the rate of the conversion of GSSG to GSH depends on the efficiency of the key enzyme GR. In this study, upon treatments and co-treatments, the intracellular GSH levels and the expressions of GR in PC12 cells were determined as oxidative stress markers (Fig. 3A and B). The results in our research demonstrated that Cd2+ (10 μM) exposure negatively affected the levels of both markers. Thus, our results were in agreement with the results found in other cell lines such as CRL-1439 normal rat liver cells (Ikediobi et al., 2004). On the other hand, CVC (100 μM) significantly boosted GSH and GR levels upon co-exposure with Cd2+ (10 μM). Thus, CVC saves PC12 cells from Cd2+-induced oxidative stress by replenishing two vital components of antioxidant defense mechanisms; the low-molecular weight thiol compound, GSH and the enzyme reducing GSSG to GSH, GR. Our results also exhibited that the acute oxidative stress posed by Cd2+ (10 μM) exposure for 48 h also caused the significant number of PC12 cell death

MT was significantly increased in co-exposed cells in comparison to only Cd2+ (10 μM) exposed cells. Thus, the results indicate that upon co-exposure, CVC increased the MT expressions in PC12 cells which increased the Cd2+ uptake by MT binding. 4. Discussion In recent times, natural antioxidants have been attentively examined since these can act as potential preventive mechanism against many toxicants to humans. In line with this, CVC, a food component possessing strong antioxidant activity, is believed to be a promising candidate (Aeschbach et al., 1994). Therefore, the objectives of this study were to define the roles of CVC on Cd2+-induced cytotoxicity and to interpret the mechanisms behind those roles through cellular and molecular perspectives. In this study, it was found that CVC effectively lessens the immensity of Cd2+-induced oxidative stress and apoptosis in PC12 cells. We also elucidated the underlying molecular mechanisms involved in the prevention of Cd2+-induced toxicity in PC12 cells for the first time. Numerous studies in a variety of cell lines suggest that Cd2+ toxicity is often associated with oxidative stress due to the over-generation of ROS causing cell-cycle degradation, deterioration of biological

Fig. 9. Western blot analysis for cytosolic cytochrome c (cyt. Cyt-c) and cytosolic AIF (cyt. AIF) in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h. (A) The representative images (cropped) of immunoblotting for cyt. Cyt-c and cyt. AIF in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. (B) Relative density for cyt. Cyt-c and cyt. AIF to β-actin in PC12 cells exposed/co-exposed with Cd2+ and CVC for 48 h. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group, respectively. 8

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Fig. 10. Effects of CVC on Cd2+ uptake in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h, measured by using ICP-MS analysis. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group, respectively.

the ROS induced DNA damages, inhibits the repair of oxidative DNA damage, affects cell cycle progression and induces apoptosis in various cell lines (e.g., HepG2 cells) (Rani et al., 2014; Skipper et al., 2016; Norbury and Zhivotovsky, 2004). In a recent study, Horvathova et al. (2007) established the inhibitory effects of CVC on H2O2 induced DNA damage in K562 cells. We measured the intact DNA density following agarose gel electrophoresis and a considerable amount of intact DNA was fragmented by exposure to Cd2+ (10 μM). However, exposure to combined Cd2+ (10 μM) and CVC (100 μM) increased the amount of intact DNA significantly (Fig. 4A and B), thereby minimizing the DNA damage. It has already been established that a leucine zipper transcription factor namely Nrf2 mediates an important signaling pathway leading to cellular protection against oxidative stress and electrophilic compounds (Loboda et al., 2016). Normally, Nrf2 is sequestered in a complex with kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. But, onset of oxidative stress disassociates and translocates Nrf2 into the nucleus to bind with antioxidant responsive element (ARE). This binding allows expressions of cytoprotective genes such as HMOX1 and NQO1 (Zhang et al., 2017; Taguchi et al., 2011). Even though the

(Fig. 1B) and increased the level of LDH activity in culture media (Fig. 2). These effects were reversed by the action of CVC (100 μM) after co-exposure. Here, it has been indicated that CVC (100 μM) can reduce the cell death by alleviating oxidative stress exerted by Cd2+ (10 μM). The oxidative stress ameliorating activity of CVC found in our research can be supported by previous findings. For instance, a recent study showed that CVC reduces oxidative stress by increasing the GSH levels against paracetamol-induced toxicity in HepG2 cells (Palabiyik et al., 2016). Cd2+ exposure induces apoptosis in a number of cell systems including PC12 cells (Rahman et al., 2017). The flow cytometry analyses in our study showed that CVC (100 μM) when co-exposed with Cd2+ (10 μM), recovered more than half of the apoptotic PC12 cells (from > 54% to about 26%) compared to only Cd2+ (10 μM) exposure (Fig. 5). One of the probable reasons behind this performance to rescue cells from apoptosis is the ameliorating effects of CVC (100 μM) on oxidative stress posed by Cd2+ (10 μM). CVC also contributed to the inhibitory effect against oxidative DNA damage, as well as enhancing effect on the cell cycle progression, through inducing antioxidant protection. Previous studies have reported that Cd2+ exposure accelerates

Fig. 11. Effects of CVC on metallothionein (MT) content in PC12 cells exposed/co-exposed with 10 μM Cd2+ and 100 μM CVC for 48 h, determined by ELISA. Error bars indicate mean ± S.E.M. (n = 3), * and # indicate significant differences (P < 0.05) from the control group and the Cd2+-exposed group, respectively. 9

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subsequently activates caspase3 and finally causes apoptosis (a process called intrinsic mitochondrial pathway) (Jiang et al., 2014; Lasfer et al., 2008). Cytochrome c is the most related and crucial hallmark molecule of mitochondrial apoptotic pathway; which after being released from mitochondria binds with Apaf-1 to form apoptosome and activates the caspase cascade (Choi et al., 2006). We found that Cd2+ (10 μM) exposure increased cytosolic cytochrome c level which was significantly lowered by the co-exposure of Cd2+ (10 μM) and CVC (100 μM) (Fig. 9A and B). Alternatively, Cd2+ can also induce apoptosis via extrinsic pathway. For instance, Pal et al. (2011) showed that Cd2+ can trigger extrinsic pathway of apoptosis by up-regulating Bid, Fas, and caspase 8 in murine hepatocytes. However, both the extrinsic and intrinsic caspase-dependent apoptotic pathways mediated by caspases 8 and 9, respectively, result in apoptosis via the cleavage and activation of the executioner caspase 3 (Wu et al., 2014). In our current research, it was depicted that Cd2+ (10 μM) significantly induces caspase 3 cleavage which is in accordance with the previous studies. However, this rise in cleaved caspase 3 level was considerably reduced by the co-exposure of Cd2+ (10 μM) and CVC (100 μM) (Fig. 8A and B). A recent finding also demonstrated that CVC provides neuroprotection on focal cerebral ischemia/reperfusion by reducing apoptosis via prohibiting caspase 3 cleavage (Yu et al., 2012). On the other hand, reports on various cell types (e.g., HEP3B cells) suggested that Cd2+ induces oxidative stress and subsequently releases AIF and endoG from the mitochondria into the cytosol causing caspase-independent apoptosis (Lemarié et al., 2004). Researchers also found AIF not only as an apoptotic executioner, but also as a survival protein (Sevriokova, 2011). Nevertheless, the translocation of AIF from mitochondria to the nucleus via cytosol is considered as a standard sign of caspase-independent apoptosis (Mao et al., 2011). We found a significant increase in the cytosolic AIF in PC12 cells after 48 h Cd2+ (10 μM) exposure which was significantly reduced by co-exposure with CVC (100 μM) (Fig. 9A and B). Therefore, it can be deduced from our findings that CVC can ameliorate both caspase-dependent and caspase-independent apoptosis upon Cd2+-induced oxidative stress. MTs are divalent, cysteine-rich small metal-binding stress-proteins ubiquitously expressed in many tissues; but expressions are induced by a variety of factors especially by metal ions such as Cd2+. It was recommended that cells synthesizing MTs are resistant, while cells not synthesizing MTs are sensitive to Cd2+ toxicity. It also have been

underlying molecular mechanism is still unclear, the inactivation of Nrf2 is necessary for the accomplishment of apoptosis (Méndez-García et al., 2019). In the current research, we found that upon co-exposure CVC (100 μM) significantly lessened the Cd2+-imposed inactivation of Nrf2 (Fig. 7A and B) and perhaps promoted the expressions of antioxidant enzymes. Additionally, the involvement of a variety of oxidative transcription factors like NFκB is responsible for achieving Cd2+induced apoptosis. It has been suggested that apoptosis due to Cd2+ toxicity in rat kidney proximal tubular epithelial cells, NRK-52E, involves the down regulation of NFκB which could facilitated oxidative stress (Xie and Shaikh, 2006). Oxidative stress also inhibits autophagy related pro-survival proteins like mTOR and Akt in the PI3/mTOR/Akt signaling pathway leading to the induction of Bax expression and suppression of Bcl-2 which ultimately activates caspase cascade to induce apoptosis (Rahman et al., 2018; Roy et al., 2014; Singh et al., 2012). As shown in Fig. 6A and B, Cd2+ (10 μM) induced downregulated expressions of NFκB, mTOR and Akt, which were considerably increased by the co-exposure of Cd2+ (10 μM) and CVC (100 μM). Furthermore, the involvement of MAPKs pathways has already been elucidated in various cell lines undergoing in vitro apoptotic as well as necrotic cell death induced by Cd2+. ERK1 belongs to MAPKs protein family which is known to be involved in cell proliferation, differentiation, and apoptosis. A contemporary research by Hu et al. (2015) has shown that Cd2+ encourages apoptosis in MG63 cells by increasing ROS and inhibiting ERK 1/2 pathway. Our results also exhibited a significant down regulation of ERK1 by Cd2+ toxicity which was meaningfully recovered by combined exposure with CVC (100 μM) (Fig. 7A and B). Again, the probable reason for ERK1 revival is that CVC could ameliorate the oxidative stress and placate the ROS activity. Cd2+ encourages mitochondrial permeability transition pore (MPTP) opening, facilitates the release of apoptogenic proteins into the cytosol and thus intensifies the occurrence of multiple apoptotic pathways via caspase-dependent (releasing cytochrome c) or via caspaseindependent (releasing AIF and Endo G) pathways. However, the activation of mitochondrial apoptotic pathways largely contributes to the manifestation of apoptotic death caused by Cd2+ toxicity (Liu et al., 2016b). Cumulative evidences from studies on a number of cell lines suggested that the Cd2+-induced oxidative stress diminishes the mitochondrial membrane potential (MMP), decreases Bcl-2/Bax ratio, releases cytochrome c into the cytosol, activates caspase 9,

Fig. 12. A schematic representation of the proposed molecular mechanism of cytoprotective and survival-enhancing effects of CVC against Cd2+-triggered oxidative stress and caspase dependent/independent apoptosis in PC12 cells. Red arrows = effects of Cd2+; green arrows = effects of CVC, CVC reduces Cd2+ toxicity by GR expression and thus enhancing the conversion of GSSG to GSH indicating the lowered occurrence of ROS. It also up-regulates the Cd2+-induced down-regulated expressions of mTOR, NFκB and Nrf2. CVC increases the expressions of Akt and ERK1 and suppresses the cleavage of caspase 3 producing less amount of c. caspase 3. Cytosolic releases of Cyt. c and AIF from mitochondria are also prohibited by CVC. Moreover, CVC reduces the DNA fragmentation and increases MT expression. The combined effect of the above phenomena exerted by CVC is the amelioration of Cd2+-induced oxidative stress and caspase dependent/independent apoptosis in PC12 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 10

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reported that MTs protect cells from oxidative species reacting with sulfhydryl groups (Ruttkay-Nedecky et al., 2013). Although the physiological functions of MTs are still debated, a number of in vivo studies showed that MTs are induced in tissues upon Cd2+ exposure and is engaged in the metabolism and detoxification through Cd2+ binding (Lu et al., 2001). Likewise, a recent in vitro study on HEK293 cell system demonstrated that MTs overexpression protected cells against Cd2+ (10 μM) toxicity (Li et al., 2005). Similarly, we found that the MTs were considerably overexpressed in PC12 cells after exposure to Cd2+ (10 μM) (Fig. 11). Surprisingly, we also observed that the MTs expression level was significantly higher in cells co-exposed to Cd2+ (10 μM) and CVC (100 μM) in comparison to that of the Cd2+ (10 μM) exposed cells. A previous study supported our finding showing that CVC can boost the overexpression of MTs in tolerogenic dendritic cells (Spiering et al., 2012). Therefore, the higher uptake of Cd2+ (Fig. 10) by PC12 cells co-exposed with Cd2+ (10 μM) and CVC (100 μM) might be explained by the overexpression of MT and probable increase in Cd2+binding by MTs. Overall, we summarized that CVC protected PC12 cells from Cd2+triggered toxicity by combating oxidative stress, increasing GSH levels, upregulating GR-expression, and reducing DNA damage; as well as, stimulating ERK-1 MAPK expression, activating expressions of Nrf2,NFκB and autophagy related pro-survival proteins-mTOR and Akt, and promoting MT overexpression(Fig. 12). Moreover, CVC effectively defended cells against both Cd2+-induced caspase-dependent and caspase-independent apoptosis through hindering caspase 3 cleavage, and prohibiting cytochrome c and AIF release into cytosol from mitochondria. Therefore, it became certain that the natural antioxidant, CVC acted as a powerful anti-oxidative and anti-apoptotic agent against Cd2+ in PC12 cells. Finally, we recommend CVC as a potential and safe therapeutic agent against the toxicity posed by the toxic heavy metal, Cd2+, in the biological system. However, further in vivo and in vitro researches are required for precise understanding of the effects, interactions and mechanism(s) of action of CVC against cadmium-induced toxicity. To add, investigations are also necessary to understand the outcome of CVC treatment on other metal or non-metal toxicants in a diverse array of biological systems.

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